Bi-specific fusion proteins for tissue repair

ABSTRACT

Bi-specific fusion proteins with therapeutic uses are provided, as well as pharmaceutical compositions comprising such fusion proteins, and methods for using such fusion proteins to repair damaged tissue. The bi-specific fusion proteins generally comprise: (a) a targeting polypeptide domain that binds to an ischemia-associated molecule; and (b) an activator domain that that detectably modulates the activity of a cellular network.

RELATED APPLICATIONS

This application claims the benefit of and priority to U.S. ProvisionalApplication Ser. No. 61/347,040, filed May 21, 2010, the entire contentof which application is herein incorporated by reference in itsentirety.

TECHNICAL FIELD

The present invention relates generally to fusion proteins that havetherapeutic uses, and more specifically to bi-specific fusion proteins,pharmaceutical compositions comprising such fusion proteins, and methodsfor using such fusion proteins to repair damaged tissue.

BACKGROUND

Myocardial infarction, commonly known as a heart attack, occurs whencoronary artery obstruction cuts off the blood supply to part of theheart. The resulting lack of oxygen causes irreversible tissue damage(necrosis and apoptosis), due to the inability of the heart tosufficiently activate endogenous regeneration programs and self-repair.Such tissue damage is a leading cause of congestive heart failure, acondition in which the heart is no longer capable of effectively pumpingblood. In the United States, there are more than a million heart attacksevery year, and nearly 5 million people are afflicted with congestiveheart failure.

There are no effective treatments for regenerating damaged cardiactissue. Current therapies for congestive heart failure focus onpreventing arrhythmia, progression of arteriosclerosis and recurrentmyocardial infarction, but do not address the underlying tissue damage.More than half of patients diagnosed with congestive heart failure diewithin five years of diagnosis.

Stem cell therapy is a potential new strategy for cardiac repair. In thelaboratory, it is possible to generate cardiac muscle cells from stemcells. This suggests that stems cells could be used to repair damagedtissue such as cardiac tissue in a patient; however, no therapeutictreatments based on such an approach are presently available. Onedifficulty that has been encountered in stem cell therapy is that oftargeting sufficient numbers of stem cells to the damaged tissue toresult in clinically significant repair.

There is, thus, a need in the art for methods for repairing orregenerating damaged tissues, including cardiac tissue, and forimproving the targeting of cells such as stem cells to facilitate tissuerepair. The present invention fulfills these needs, and provides otherrelated advantages.

SUMMARY OF THE INVENTION

The present invention provides bi-specific fusion proteins, nucleic acidmolecules encoding bi-specific fusion proteins and therapeutic methodsthat employ such bi-specific fusion proteins.

In certain aspects, the present invention provides bi-specific fusionproteins that comprise: (a) a targeting domain having a bindingspecificity to an ischemia-associated molecule; and (b) an activatordomain having a binding specificity to a growth factor receptor orcytokine receptor, wherein upon exposure of the activator domain to thegrowth factor receptor or cytokine receptor, the activator domain bindsthe growth factor receptor or cytokine receptor so as to modulateregeneration of a cardiac tissue.

In some embodiments, the bi-specific protein comprises: (a) a targetingpolypeptide domain that binds to an ischemia-associated molecule with aK_(d) (i.e., said binding exhibits a K_(d)) ranging from 10⁻⁶ to 10⁻¹² Mor better; and (b) an activator domain that that detectably modulatesthe activity of a cellular network (e.g., detectably modulatesactivation of a growth factor receptor or cytokine receptor). In certainembodiments, the targeting polypeptide domain binds to theischemia-associated molecule with a K_(d) ranging from 10⁻⁷ to 10⁻¹² Mor better, or ranging from 10⁻⁸ to 10⁻¹² M or better. In furtherembodiments, the K_(d) is determined using a biosensor, e.g., by surfaceplasmon resonance or resonant mirror analysis.

In addition to components (a) and (b), above, certain bi-specific fusionproteins provided herein further comprise: (c) a polypeptide linkerwherein the polypeptide linker extends the half life of the bi-specificfusion protein. In some embodiments, the targeting domain is at theN-terminus or at the C-terminus of the activator domain. In otherembodiments, the polypeptide linkers at the N-terminus or at theC-terminus of the targeting domain. In some embodiments, the targetingdomain is at the amino terminus of the fusion protein and the activatordomain is at the carboxy terminus of the fusion protein. Yet in otherembodiments, the targeting domain is at the carboxy terminus of thefusion protein and the activator domain is at the amino terminus of thefusion protein. In some embodiments, the polypeptide linker has twotermini, an N-terminus and a C-terminus, that is joined at one terminusvia a peptide bond to the targeting polypeptide domain and is joined atthe other terminus via a peptide bond to the activator domain. Incertain such embodiments, the targeting peptide is linked to theN-terminus of the linker and the activator domain is linked to theC-terminus of the linker. In other such embodiments, the targetingpeptide is linked to the C-terminus of the linker and the activatordomain is linked to the N-terminus of the linker. In certainembodiments, the linker is non-immunogenic in humans (e.g., a humanserum protein or derivative thereof). Representative such linkerscomprise at least 100 consecutive amino acids that are at least 80%identical to a serum albumin amino acid sequence, such as a humanalpha-fetoprotein sequence. In certain embodiments, the linker comprisesor has an amino acid sequence recited in any one of SEQ ID NOs: 10-29.

In some embodiments, the bi-specific fusion protein comprises (a) atargeting domain having a binding specificity to an ischemia-associatedmolecule; (b) an activator domain that detectably modulates activationof a receptor; and (c) a polypeptide linker, wherein the polypeptidelinker extends the half life of the bi-specific fusion protein.

In some embodiments, the bi-specific fusion protein comprises (a) atargeting domain having a binding specificity to a target molecule; (b)an activator domain having a binding specificity to a receptor, whereinupon exposure of the activator domain to the receptor, the activatordomain binds the receptor so as to modulate activation of the receptor;and (c) a polypeptide linker, wherein the polypeptide linker extends thehalf life of the bi-specific fusion protein.

In some embodiments, the bi-specific protein comprises (a) a targetingdomain having a binding specificity to a tissue-associated molecule; and(b) an activator domain having a binding specificity to a moleculeassociated with the surface of a cell in the tissue, wherein uponexposure of the activator domain to surface-associated molecule, theactivator domain binds the membrane-associated molecule so as tomodulate regeneration of the tissue, wherein the targeting domain andthe activator domain are linked via a linker, and wherein the linkerextends the half life of the bi-specific fusion protein.

In some embodiments, the bi-specific fusion protein comprises (a) atargeting domain having a binding specificity to a target molecule; (b)an activator domain having a binding specificity to a receptor, whereinupon exposure of the activator domain to the receptor, the activatordomain binds the receptor so as to modulate tissue regeneration; and (c)a polypeptide linker, wherein the polypeptide linker extends the halflife of the bi-specific fusion protein.

In some embodiments, the targeting domain binds to the molecule with adissociation constant Kd ranging from 10⁻⁶ M to 10⁻¹² M. In someembodiments, the targeting domain binds to a molecule selected from thegroup of mysosin, cardiac myosin, DNA, phosphatidylserine, collagen, orextracellular matrix proteins. For example, the targeting domain can beselected from the group of annexin, anti-myosin antibody, anti-DNA scFv,variants thereof, fragments thereof, and combinations thereof. In someembodiments, the scFv antibody has a sequence recited in any one of SEQID NOs: 1, 2, or 30. In some embodiments, annexin has a sequence recitedin SEQ ID NO: 31.

In some embodiments, the activator domain binds specifically to a growthfactor receptor or cytokine receptor. For example, the activator domainis selected from the group consisting of hepatocyte growth factor,vascular endothelial growth factor, fibroblast growth factor,neuregulin/heregulin, variant thereof, and portion thereof.

In other embodiments, the bi-specific fusion proteins comprises (a) aleader polypeptide that comprises a sequence recited in SEQ ID NO:41 or42; (b) a targeting polypeptide domain that binds to anischemia-associated molecule, said binding exhibiting a K_(d) rangingfrom 10⁻⁶ to 10⁻¹² M or better; (c) a short connector polypeptide thatcomprises the sequence -Gly-Ala- or -Ala-Ser-; (d) a HSA polypeptidethat comprises a sequence recited in any one of SEQ ID NOs:10, 12, 14-29and 45); (e) a short connector polypeptide that comprises the sequence-Leu-Gln- or -Thr-Gly-; (f) an activator domain that that detectablymodulates the activity of a cellular network; and (g) ahexahistidine-comprising polypeptide.

It will be apparent that the above components may be present in thebi-specific fusion protein in the order recited or in a different order(e.g., the locations of the targeting polypeptide domain and activatordomain may be switched). Within certain such bi-specific fusionproteins, the targeting polypeptide domain comprises a sequence recitedin SEQ ID NO:1, 2, 30 or 31; the HSA polypeptide comprises the sequencerecited in SEQ ID NO:45; the activator domain comprises a sequencerecited in any one of SEQ ID NOs: 32-40; and thehexahistidine-comprising polypeptide has a sequence recited in SEQ IDNO:43 or 44.

In certain embodiments of the bi-specific fusion proteins describedabove, the ischemia-associated molecule is a DNA molecule, myosin (e.g.,a myosin subtype such as cardiomyosin) or phosphatidyl serine.

In certain embodiments of the bi-specific fusion proteins describedabove, the targeting polypeptide comprises an antibody variable region.In certain such embodiments, the targeting polypeptide comprises a scFvantibody. Representative such scFv antibodies comprise or have asequence recited in SEQ ID NO:1 or SEQ ID NO:2.

In certain embodiments of the bi-specific fusion proteins describedabove, the activator domain is a growth factor polypeptide. Withincertain such embodiments, the growth factor polypeptide binds to areceptor for IGF or HGF (e.g., the growth factor polypeptide comprisesor has an amino acid sequence recited in any one of SEQ ID NOs:3-9).

The bi-specific binding agents provided herein are not necessarilylimited to two binding specificities. In certain embodiments, inaddition to the targeting domain, the bi-specific fusion proteincomprises two or more activator domains that are linked directly orindirectly via peptide bonds and are selected from growth factorpolypeptides and cytokine polypeptides.

In other aspects, the present invention provides pharmaceuticalcompositions, comprising a bi-specific fusion protein as described abovein combination with a physiologically acceptable carrier.

Within still further aspects, methods are provided for treatingpathological tissue damage in a patient, comprising administering apharmaceutical composition to a patient suffering from pathologicaltissue damage, and thereby decreasing pathological tissue damage in thepatient. In certain embodiments, the pathological tissue damage is hearttissue damage associated with myocardial infarction. In otherembodiments, the pathological tissue damage is kidney tissue damage.

In some embodiments, methods are provided for promoting tissueregeneration in a patient. The methods comprise (a) providing abi-specific fusion protein comprising (i) a targeting domain having abinding specificity to an ischemia-associated molecule; and (ii) anactivator domain having a binding specificity to growth factor receptoror cytokine receptor; and (b) administering in a patient in need thereofa therapeutically effective amount of the bi-specific fusion proteinwhereby the targeting domain specifically binds to theischemia-associated molecule thereby targeting the bi-specific fusionprotein to a tissue and whereby upon exposure of the activator domain tothe growth factor receptor or cytokine receptor, the activator domainspecifically activates the growth factor receptor or cytokine receptorso as to promote tissue regeneration. In some embodiments, the methodscomprise (a) providing a bi-specific fusion protein comprising (i) atargeting domain having a binding specificity to a target molecule; (ii)an activator domain having a binding specificity to a receptor; (iii) apolypeptide linker, wherein the polypeptide linker extends the half lifeof the bi-specific fusion protein; and (b) administering in a patient inneed thereof a therapeutically effective amount of the bi-specificfusion protein whereby the targeting domain specifically binds to thetarget molecule thereby targeting the bi-specific fusion protein to afirst cell and whereby upon exposure of the activator domain to thegrowth factor receptor, the activator domain specifically activates thereceptor of a second cell of a tissue so as to promote tissueregeneration.

In certain embodiments, such methods further comprise the administrationof stem cells to the patient. In some embodiments, upon administrationof the bi-specific protein, the bi-specific protein prevents celldamage, increases survival, promotes cell growth, promotes motility ofstem cells, recruits stem cells, promotes differentiation of stem cells.

Also provided herein are nucleic acid molecules encoding a bi-specificfusion protein as described above. In certain embodiments, the nucleicacid molecule is DNA, and the DNA further comprises transcriptional andtranslational regulatory sequences operably linked to the bi-specificfusion protein coding sequence, such that transcription and translationof the coding sequence occurs in at least one eukaryotic cell type.

These and other aspects of the present invention will become apparentupon reference to the following detailed description.

DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO:1 is the amino acid sequence of the anti-DNA scFv SV-1.

SEQ ID NO:2 is the amino acid sequence of the anti-DNA scFv SV-22.

SEQ ID NO:3 is the amino acid sequence of a growth factor polypeptidecorresponding to wild type human IGF-I (mature form).

SEQ ID NO:4 is the amino acid sequence of a growth factor polypeptidecorresponding to human IGF-1 with D12A substitution.

SEQ ID NO:5 is the amino acid sequence of a growth factor polypeptidecorresponding to human IGF-1 with E9A substitution.

SEQ ID NO:6 is the amino acid sequence of a growth factor polypeptidecorresponding to human HGF alpha chain N-K1 domain.

SEQ ID NO:7 is the amino acid sequence of a growth factor polypeptidecorresponding to human HGF alpha chain K1 domain.

SEQ ID NO:8 is the amino acid sequence of a growth factor polypeptidecorresponding to human HGF alpha chain N-K2 fusion.

SEQ ID NO:9 is the amino acid sequence of a growth factor polypeptidecorresponding to human HGF alpha chain K2 domain.

SEQ ID NO:10 is the amino acid sequence of a human serum albumin (HSA)linker with C34S and N503Q substitutions.

SEQ ID NO:11 is the nucleic acid sequence of an HSA linker with C34S andN503Q substitutions.

SEQ ID NO:12 is the amino acid sequence of HSA.

SEQ ID NO:13 is the nucleic acid sequence of HSA.

SEQ ID NO:14 is the amino acid sequence of an HSA linker with C34S andN503Q substitutions and a polypeptide connector.

SEQ ID NO:15 is the amino acid sequence of an HSA linker with C34S andN503Q substitutions and a polypeptide connector.

SEQ ID NO:16 is the amino acid sequence of an HSA linker with C34S andN503Q substitutions and a polypeptide connector.

SEQ ID NO:17 is the amino acid sequence of an HSA linker with C34S andN503Q substitutions and a polypeptide connector.

SEQ ID NO:18 is the amino acid sequence of an HSA linker with C34S andN503Q substitutions and a polypeptide connector.

SEQ ID NO:19 is the amino acid sequence of an HSA linker with apolypeptide connector.

SEQ ID NO:20 is the amino acid sequence of an HSA linker with apolypeptide connector.

SEQ ID NO:21 is the amino acid sequence of an HSA linker with apolypeptide connector.

SEQ ID NO:22 is the amino acid sequence of an HSA linker with apolypeptide connector.

SEQ ID NO:23 is the amino acid sequence of an HSA linker with apolypeptide connector.

SEQ ID NO:24 is the amino acid sequence of an HSA linker with C34Ssubstitution, domain I.

SEQ ID NO:25 is the amino acid sequence of an HSA linker, domain II.

SEQ ID NO:26 is the amino acid sequence of an HSA linker with N503Qsubstitution, domain III.

SEQ ID NO:27 is the amino acid sequence of an HSA linker, domain I.

SEQ ID NO:28 is the amino acid sequence of an HSA linker, domain II.

SEQ ID NO:29 is the amino acid sequence of human alpha-fetoprotein.

SEQ ID NO:30 is the amino acid sequence of the anti-phosphatidylserinescFv PS4A7.

SEQ ID NO:31 is the amino acid sequence of human annexin V.

SEQ ID NO:32 is an amino acid sequence of a growth factor polypeptidecorresponding to human HGF alpha chain N-K1 domain.

SEQ ID NO:33 is an amino acid sequence of a growth factor polypeptidecorresponding to human HGF alpha chain K1 domain.

SEQ ID NO:34 is an amino acid sequence of a growth factor polypeptidecorresponding to human HGF alpha chain N-K2 domain.

SEQ ID NO:35 is an amino acid sequence of a growth factor polypeptidecorresponding to human HGF alpha chain K2 domain.

SEQ ID NO:36 is an amino acid sequence of a growth factor polypeptidecorresponding to human VEGF alpha monomer.

SEQ ID NO:37 is an amino acid sequence of a growth factor polypeptidecorresponding to human VEGF alpha dimer.

SEQ ID NO:38 is an amino acid sequence of a growth factor polypeptidecorresponding to human FGF2.

SEQ ID NO:39 is an amino acid sequence of a growth factor polypeptidecorresponding to human NRG1 alpha, EGF-like domain.

SEQ ID NO:40 is an amino acid sequence of a growth factor polypeptidecorresponding to human NRG1 alpha, full sequence.

SEQ ID NO:41 is an amino acid sequence of a bi-specific fusion proteinleader polypeptide.

SEQ ID NO:42 is an amino acid sequence of a bi-specific fusion proteinleader polypeptide.

SEQ ID NO:43 is an amino acid sequence of a C-terminalhexahistidine-comprising polypeptide.

SEQ ID NO:44 is an amino acid sequence of a C-terminalhexahistidine-comprising polypeptide.

SEQ ID NO:45 is an amino acid sequence of a HSA linker.

SEQ ID NO:46 is an amino acid sequence of a HSA linker with N-terminaland C-terminal short connector polypeptides.

SEQ ID NO:47 is an amino acid sequence of a HSA linker with N-terminaland C-terminal short connector polypeptides.

SEQ ID NO:48 is an amino acid sequence of a HSA linker with N-terminaland C-terminal short connector polypeptides.

SEQ ID NO:49 is an amino acid sequence of a HSA linker with N-terminaland C-terminal short connector polypeptides.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to bi-specific fusion proteins thatcomprise: (1) a targeting polypeptide domain that binds to anischemia-associated molecule; and (2) an activator domain, such as agrowth factor polypeptide or a cytokine polypeptide. In certainembodiments, the bi-specific fusion protein further comprises: (3) apolypeptide linker having two termini, an N-terminus and a C-terminus,that is joined at one terminus via a peptide bond to the targetingpolypeptide domain and is joined at the other terminus via a peptidebond to the activator domain. Such bi-specific fusion proteins find use,for example, in recruiting cells that express one or more growth factorand/or cytokine (e.g., chemokine) receptors (e.g., stem cells,progenitor cells or immune system cells) to tissue following an ischemicevent (e.g., to damaged cells). In vivo, the administration of suchbi-specific fusion proteins may be used to facilitate repair orregeneration of damaged tissue.

The term “polypeptide” is used herein to refer to a molecule thatconsists of multiple amino acid residues linked by peptide bonds. Thisterm carries no implication as to the number of amino acid residues solinked.

The term “bi-specific” as used herein, refers to the ability of thefusion protein to interact with two different ligands: anischemia-associated molecule (bound by the targeting polypeptide domain)and a receptor for the activator domain. The binding properties of thetargeting polypeptide domain and the activator domain are discussed inmore detail below.

An “ischemia-associated molecule” is any molecule that is detected at alevel that is significantly higher (e.g., at least 2-fold higher)following ischemia or hypoxia. Any suitable binding assay may be used toidentify ischemia-associated molecules, including those provided herein.The increased level of molecule that is detected may be the result ofupregulation or decreased turnover, or may be due to increasedaccessibility (e.g., resulting from cell damage). In certainembodiments, the ischemia-associated molecule is detected in a cell ofpost-ischemic tissue at a significantly higher level (e.g., at least2-fold higher) than in a cell of the same tissue that has not undergonean ischemic event (i.e., the molecule is specific to or enriched in thepost-ischemic tissue). In further embodiments, the ischemia-associatedmolecule is associated with cell damage (i.e., the molecule is detectedat a significantly higher level in cells that are damaged than inundamaged cells of the same type).

Certain ischemia-associated molecules are enriched (2 fold or higher) inthe heart after an ischemic event (or in a model system that is used tomimic ischemia in the heart). Such molecules include, for example,molecules that are exposed on myocytes or other cardiac cells thatundergo necrosis (such as DNA) or apoptosis (e.g., phosphatidylserine)or molecules that are enriched in scarred heart tissue, such as collagen(collagen I, III), myosin (including the cell type-specific subtypesthereof), or other extracellular matrix proteins that are enriched inpost ischemic hearts. Such molecules can be identified on the basis ofenrichment following ischemia-reperfusion in vivo or in simulatedischemia-reperfusion in vitro, or following exposure to conditions suchas hypoxia, decreased ATP, increased reactive oxygen species (ROS) ornitric oxide synthase (NOS) production, or serum starvation of cellscultured in vitro.

The Targeting Polypeptide Domain

Binding to the ischemia-associated molecule is mediated by the targetingpolypeptide domain. This domain may be any polypeptide sequence thatserves this function; in preferred embodiments, the targetingpolypeptide domain comprises one or more antibody variable regions.

As used herein, an “antibody” is a protein consisting of one or morepolypeptides substantially encoded by immunoglobulin genes. A typicalantibody is a tetramer that is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). “V_(L)” and V_(H)″ refer to these lightand heavy chains respectively. An “antibody variable region” is anN-terminal region of an antibody variable chain (V_(L) or V_(H))comprising amino acid residues that are primarily responsible forantigen recognition. Those of ordinary skill in the art are readily ableto identify an antibody variable region and to determine the minimumsize needed to confer antigen recognition. Typically, an antibodyvariable region comprises at least 70 amino acid residues, and morecommonly at least 100 amino acid residues. A polypeptide that comprisesan antibody variable region may (but need not) further comprise otherlight and/or heavy chain sequences, and may (but need not) furthercomprise sequences that are not antibody-derived. It will be apparentthat the sequence of an antibody variable region may benaturally-occurring, or may be modified using standard techniques,provided that the function (antigen recognition) is retained. Certainpolypeptides that comprise an antibody variable region are single chainantibodies (antibodies that exist as a single polypeptide chain), morepreferably single chain Fv antibodies (scFv) in which a variable heavychain region and a variable light chain region are joined together(directly or through a peptide linker) to form a continuous polypeptide.The scFv antibody may be chemically synthesized or may be expressed froma nucleic acid including V_(H)- and V_(L)-encoding sequences eitherjoined directly or joined by a peptide-encoding linker.

“Binding” indicates that an antibody exhibits substantial affinity for aspecific antigen (e.g., an ischemia-associated molecule) and is said tooccur when the fusion protein (or the targeting polypeptide domainthereof) has a substantial affinity for the target antigen and isselective in that it does not exhibit significant cross-reactivity withother antigens. Preferred substantial binding includes binding with adissociation constant (K_(d)) of 10⁻⁶, 10⁻⁷, 10⁻⁸, 10⁻⁹, 10⁻¹⁰, 10⁻¹¹,10⁻¹² M or better. The K_(d) of an antibody-antigen interactionindicates the concentration of antibody (expressed as molarity) at which50% of antibody and antigen molecules are bound together atthermodynamic equilibrium. Thus, at a suitable fixed antigenconcentration, 50% of a higher (i.e., stronger) affinity antibody willbind antigen molecules at a lower antibody concentration than would berequired to achieve the same percent binding with a lower affinityantibody. K_(d) is also the a ratio of the kinetic on and off rates(k_(on) and k_(off)); i.e., K_(d)=k_(off)/k_(on). Thus, a lower K_(d)value indicates a higher (stronger) affinity. As used herein, “better”affinities are stronger affinities, and are identified by dissociationconstants of lower numeric value than their comparators, with a K_(d) of10⁻¹° M being of lower numeric value and therefore representing a betteraffinity than a K_(d) of 10⁻⁹M. Affinities better (i.e., with a lowerK_(d) value and therefore stronger) than 10⁻⁷M, preferably better than10⁻⁸M, are generally preferred. Values intermediate to those set forthherein are also contemplated, and preferred binding affinity can beindicated as a range of dissociation constants, for example preferredbinding affinities for antibodies disclosed herein are represented byK_(d) values ranging from 10⁻⁶ to 10⁻¹² M (i.e., micromolar topicomolar), preferably 10⁻⁷ to 10⁻¹² M, more preferably 10⁻⁸ to 10⁻¹² Mor better. An antibody that “does not exhibit significantcross-reactivity” is one that will not appreciably bind to an off-targetantigen. For example, in one embodiment, an antibody that specificallyand selectively binds to Annexin V will exhibit at least a two, andpreferably three, or four or more orders of magnitude better bindingaffinity (i.e., binding exhibiting a two, three, or four or more ordersof magnitude lower K_(d) value) for Annexin V than for Annexin moleculesother than Annexin V or for non-Annexin proteins or peptides. Bindingaffinity and selectivity can be determined using any art-recognizedmethods for determining such characteristics, including, for example,using Scatchard analysis and/or competitive (competition) bindingassays.

Binding may be assessed, and K_(d) values determined, using any of avariety of techniques that are well known in the art. For example,binding to an ischemia-associated DNA molecule is commonly assessed bycoating an appropriate solid support (e.g., beads, ELISA plate orBIACORE chip) with target DNA fragments. For a targeting polypeptidedomain that binds to any sequence of DNA, DNA fragments (single ordouble-stranded) of 10 base pairs or larger are immobilized on the solidsubstrate. For a targeting polypeptide domain that binds to a specificsequence or DNA complex (e.g., DNA-histone complex) the appropriatecorresponding target is immobilized. Prior to adding theischemia-associated molecule, non-specific binding sites for protein areblocked with BSA, milk, or any other appropriate blocker. Uncoated wellsor wells coated with a non-target molecule serve as specificitycontrols. Increasing concentrations of the bi-specific fusion protein(or targeting polypeptide domain) are incubated with target-coatedsubstrate or control substrate. A fusion protein or domain that does notbind to the target is also tested as a specificity control. Targetspecific, dose-dependent binding of the bi-specific fusion protein (ortargeting polypeptide domain) is then assessed by measuring the amountof bi-specific fusion protein (or targeting polypeptide domain) bindingto target versus controls as a function of increasing dose usingstandard protocols corresponding to the solid support and bindingtechnology being used. Representative such protocols include thosedescribed in Wassaf et al., Anal. Biochem. 351(2):241-53 (2006); Epub2006 Feb. 10 (BIACORE); and Murray and Brown, J. Immunol. Methods.127(1):25-8 (1990) (ELISA). In addition, studies that vary the amount ofimmobilized target molecule or that include increasing levels of solubletarget molecule as a competitor may also be performed to monitor bindingand specificity.

The binding affinity and kinetic on and off rates for binding to thetarget molecule are measured using standard techniques and compared toother negative control molecules (e.g., fusion protein with irrelevanttargeting polypeptide or fusion protein lacking a targeting polypeptide)and positive control molecules (e.g., parental antibody that targets theischemia-associated molecule, or other antibodies or antibody fragmentsthat are known to bind to the ischemia-associated molecule).

In certain embodiments, the K_(d) is determined using a biosensor (e.g.,by surface Plasmon resonance (BIAcore) or resonant mirror analysis(IAsys)). Such determinations may be performed as described by Hefta etal., Measuring Affinity Using Biosensors, in “Antibody Engineering: APractical Approach,” McCafferty et al. (eds), pp. 99-116 (OxfordUniversity Press, 1996), and references cited therein. Briefly, kineticon and off rates (k_(on) and k_(off)) are determined using a sensor chipto which the ischemia-associated molecule has been coupled. To evaluateassociation (k_(on)), solutions of different concentrations ofbi-specific fusion protein (or targeting polypeptide domain) flow acrossthe chip while binding is monitored using mass sensitive detection.Using the BIAcore system (GE Healthcare; Piscataway, N.J.), k_(on) isthe slope of the plot of dR/dt versus R, where R is the signal observed.Following binding, dissociation is observed by passing a buffer solutionacross the chip, and k_(off) is determined in an analogous fashion.K_(d) is then calculated using the equation:K _(d) =k _(off) /k _(on)

In the context of the present invention, a bi-specific fusion proteinbinds to the ischemia-associated molecule if it binds with a K_(d) ofless than 10⁻⁸ M, preferably less than 10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M or 10⁻¹⁰M. In addition, the binding of the bi-specific fusion protein to theischemia-associated molecule in this assay is significantly higher(e.g., at least 2-, 10- or 100-fold higher) than binding of thebi-specific fusion protein to negative controls. Preferably, binding tothe immobilized target can also be competed using excess soluble target.

As noted above, certain ischemia-associated molecules are specific to(or enriched in) damaged cells. Binding to damaged cells is convenientlydemonstrated in vitro using cultured cells that are exposed toconditions that induce necrosis or apoptosis. For example, necrosis canbe induced in cultured cardiomyocytes by simulated ischemia/reperfusion,and monitored using a LDH release assay, or trypan blue assay followedby subtraction of the number of cells undergoing apoptosis, essentiallyas described in Shan et al., Am. J. Physiol. Cell. Physiol. 294:833-841(2008). This assay quantitates the total dead cells and the differencebetween the total and the number of apoptotic cells is attributed tonecrosis, as discussed in more detail below. Conditions that induceapoptosis include exposure to H₂O₂, and apoptosis can be monitored usingany of a variety of techniques known in the art including, for example,annexin V (SEQ ID No. 31) binding cleavage of target peptide sequencesby known caspases that are activated by apoptosis, or DNA laddering(measured by TUNEL assay, essentially as described in Kuramochi, J.Biol. Chem. 279(49): 51141-47 (2004)). Binding to the cells undergoingnecrosis or apoptosis may be assessed by adding fluorescently labeledbi-specific fusion protein (or targeting polypeptide domain) orappropriate control proteins to cells following the induction ofapoptosis or necrosis. After incubation of the proteins with the cellsfor times ranging from a few minutes to one day, the cells are washedand then the cell-bound fluorescence is measured usingimmunofluorescence, flow cytometry, or similar techniques.Alternatively, other methods of detecting the bound hi-specific fusionprotein (or targeting polypeptide domain) may be used, includingradiolabeling or using enzymes conjugated to the hi-specific fusionprotein (or targeting polypeptide domain) or to antibodies that bind tothe fusion protein (or targeting polypeptide domain), which is commonpractice in ELISA protocols. The hi-specific fusion protein (ortargeting polypeptide domain) binds to target cells if significantlyhigher (e.g., 2-fold higher) binding to cells following ischemia (e.g.,cells undergoing necrosis or apoptosis) is detected, as compared tocells that have not experienced an ischemic event (e.g., cells notundergoing apoptosis or necrosis).

In vivo targeting may be demonstrated by inducing ischemia in an animalmodel and comparing the level of administered bi-specific fusion protein(or targeting polypeptide domain) in a target tissue before and afterischemia. In vivo targeting to damaged cells may be demonstrated byinducing tissue damage in an animal model, administering the bi-specificfusion protein (or targeting polypeptide domain), and comparing thelevel of bi-specific fusion protein (or targeting polypeptide domain) indamaged versus undamaged cells. In one embodiment, the bi-specificfusion proteins are designed to target areas of tissue damage followingischemia-reperfusion injury. In such a case, demonstration of in vivotargeting may be accomplished by inducing tissue damage, preferably by amethod that causes ischemia followed by re-establishment of bloodsupply. Numerous methods are available to do this in different tissues.For example, blood flow to the hindlimb of the mouse can be transientlyblocked with a simple tourniquet. Alternatively, temporary clamp on theartery leading into the kidney can be employed. Ischemia-reperfusioninjury can be induced in the heart through temporary blockage of thecoronary artery as demonstrated in mice, rats, dogs, and pigs.Representative methods for inducing tissue damage in an animal model aresummarized in Table 1.

TABLE 1 Representative Methods used to Induce Ischemia-ReperfusionDamage Organ or tissue Methods used to induce damage Reference HeartMouse: left anterior descending Dumont et al., Circulation arteryclamped for up to 30 102(13): 1564-8 (2000) minutes followed byreperfusion Davis, Proc. Natl. Acad. Sci. Rat: coronary artery ligationUSA 23: 103(21): 8155-60 (2006) Kidney Mouse: Renal artery clamped Chenet al., FASEB J. 4(12): with pediatric suture for 1-6 hrs 3033-39 (1990)Liver Dog: The hepatic pedicle and Miranda et al., Braz. J. Med. hepaticartery (close to the celiac Biol. Res 40(6): 857-65 (2007) artery) werecross-clamped with Kobayashi et al., World J. vascular clamps.Gastroentero1. 13(25): Pig: Details in reference 3487-92 (2007) Hind-Zbinden et al., Am. J. Physiol. limb Heart Circ. Physiol. 292:H1891-H1897 (2007)

Animal models for ischemia-reperfusion injury are further detailed inthe following references:

-   Greenberg et al., Chapter 7. Mouse models of ischemic angiogenesis    and ischemia-reperfusion injury. Methods Enzymol. 444:159-74 (2008).-   Chimenti et al., Myocardial infarction: animal models. Methods Mol.    Med. 98:217-26 (2004).-   Black S C, In vivo models of myocardial ischemia and reperfusion    injury: application to drug discovery and evaluation. J. Pharmacol.    Toxicol. Methods 43(2):153-67 (2000).

The specificity of targeting can be established by comparing thebi-specific fusion protein (or targeting polypeptide domain) depositionin the clamped versus unclamped kidney as shown in Chen et al., FASEB J.4(12): 3033-39 (1990), or in the treated versus untreated hindlimb asshown in Zbinden et al., Am. J. Physiol. Heart Circ. Physiol. 292:H1891-H1897 (2007), using radiolabeled bi-specific fusion protein (ortargeting polypeptide domain). Alternatively, bi-specific fusion protein(or targeting polypeptide domain) can be detected in homogenized tissueusing ELISA, or can be imaged in real time using bi-specific fusionprotein (or targeting polypeptide domain) labeled with the appropriatemetal for imaging (e.g., Tc99, Y or Gd). Specific deposition in thedamaged area of the heart can be measured as described in Dumont et al.,Circulation 102(13):1564-8 (2000). Representative methods fordemonstrating targeting of proteins to damaged tissue are shown in Table2.

TABLE 2 Demonstration of Targeting to Damaged Tissue Damaged organ ortissue Methods used to demonstrate targeted targeted delivery ReferenceHeart Humans: Tc99 labeling of Annexin Hofstra et al., The V followed byimaging in humans Lancet 356(9225): using SPECT in patients with 209-12(2000) myocardial infarction followed by reperfusion attempts viaangioplasty or thrombolysis Heart Mouse: Fluorescent labeling of Dumontet al., Annexin V in murine model of Circulation ischemia reperfusionwith 102(13): 1564-8 distribution in the myocardium (2000) detectedhistologically Heart Humans: Tc99 labeling of Annexin Hofstra et al.,The V followed by imaging in humans Lancet 356(9225): using SPECT inpatients undergoing 209-12 (2000) cardiac transplant rejection HeartMouse: Fluorescently-labeled growth Urbanek, Proc. Natl. factor imagedin heart tissue using Acad. Sci. USA 102 confocal microscopy (24):8692-97 (2005) Damaged Radiographs of clamped versus Chen et al., FASEBJ. kidney unclamped kidney 4(12): 3033-9 (1990) targetedMicroautoradiographs to show using localization to specific cellularradiolabeled structures in the kidney antibody Imaging of whole mouseusing to DNA I131-labeled antibody to DNA (versus labeled control)Biodistribution of I125-labeled antibody to show deposition innon-target tissues

As noted above, certain targeting polypeptide domains comprise a scFvantibody that binds to the ischemia-associated molecule. Representativesuch scFv antibodies comprise or have the sequences provided herein asSEQ ID NOs: 1, 2, and 30.

It will be apparent that functionally related antibodies may also, oralternatively, be used as a targeting polypeptide domain. Antibodiesinteract with target antigens predominantly through amino acid residuesthat are located in the six heavy and light chain complementaritydetermining regions (CDRs). For this reason, the amino acid sequenceswithin CDRs are more diverse between individual antibodies thansequences outside of CDRs. Because CDR sequences are responsible formost antibody-antigen interactions, it is possible to generate modifiedantibodies that mimic the properties of an original antibody bycombining CDR sequences from one antibody with framework sequences froma different antibody. Such framework sequences can be obtained frompublic DNA databases that include germline antibody gene sequences.

Thus, one or more CDRs of a targeting polypeptide domain sequenceprovided herein, can be used to create functionally related antibodiesthat retain the binding characteristics of the original targetingpolypeptide domain. In one embodiment, one or more CDR regions selectedfrom SEQ ID NOs: 1, 2, and 30, is combined recombinantly with knownhuman framework regions and CDRs to create additional,recombinantly-engineered, targeting polypeptide domains. The heavy andlight chain variable framework regions can be derived from the same ordifferent antibody sequences. CDR regions are readily identified usingalignments with known sequences in databases such as Vbase and IMGT. Theresulting targeting polypeptide domains share one or more CDRs with thetargeting polypeptide domains of SEQ ID NOs: 1, 2, and 30; in certainembodiments, the targeting polypeptide domain comprises at least one CDRof a sequence as recited in SEQ ID NO: 1, 2, or 30.

It is well known in the art that antibody heavy and light chain CDR3domains play a particularly important role in the bindingspecificity/affinity of an antibody for an antigen. Accordingly, incertain embodiments, antibodies are generated that include the heavyand/or light chain CDR3s of the particular antibodies described herein.The antibodies can further include the heavy and/or light chain CDR1and/or CDR2s of the antibodies disclosed herein.

The CDR 1, 2, and/or 3 regions of the engineered antibodies describedabove can comprise the exact amino acid sequence(s) as those disclosedherein. However, the ordinarily skilled artisan will appreciate thatsome deviation from the exact CDR sequences may be possible,particularly for CDR1 and CDR2 sequences, which can tolerate morevariation than CDR3 sequences without altering epitope specificity (suchdeviations are, e.g., conservative amino acid substitutions).Accordingly, in another embodiment, the engineered antibody may becomposed of one or more CDR1s and CDR2s that are, for example, 90%, 95%,98%, 99% or 99.5% identical to the corresponding CDRs of an antibodynamed herein.

In another embodiment, one or more residues of a CDR may be altered tomodify binding to achieve a more favored on-rate of binding. Using thisstrategy, an antibody having ultra high binding affinity (e.g.,K_(d)=10⁻¹⁰ or less) can be achieved. Affinity maturation techniques,well known in the art, can be used to alter the CDR region(s) followedby screening of the resultant binding molecules for the desired changein binding. Accordingly, as CDR(s) are altered, changes in bindingaffinity as well as immunogenicity can be monitored and scored such thatan antibody optimized for the best combined binding and lowimmunogenicity are achieved.

Modifications can also be made within one or more of the framework orjoining regions of the heavy and/or the light chain variable regions ofan antibody, so long as antigen binding affinity subsequent to thesemodifications is not substantially diminished.

The Activator Domain

The activator domain is any polypeptide that detectably modulates theactivity of a cellular network; certain activator domains are growthfactor polypeptides or cytokine polypeptides (e.g., a chemokinepolypeptide). It will be apparent that such modulation may be anincrease or a decrease in the activity of the cellular network. A growthfactor polypeptide detectably modulates activation of a growth factorreceptor (such as HGF or IGF receptor). Certain such polypeptides arewild-type hepatocyte growth factor (HGF) or HGF alpha chain (e.g.,GENBANK accession number P14210), or derivatives thereof that retain atleast 10% of wild-type biological activity, as determined by measuringactivation of the corresponding growth factor receptor in appropriatetarget cells. Activation may be assessed, for example, by measuringphosphorylation of receptor kinase or downstream proteins, such as AKT,essentially as described by Nishi et al., Proc. Natl. Acad. Sci. USA95:7018-7023 (1998). MTT and CTG assays known in the art may also beused. Representative growth factor polypeptides have a sequence asrecited in SEQ ID NO:3-9 or 32-40, herein. As discussed above for thetargeting polypeptide domain, activator domains that share one or moreCDRs with the activator domains of SEQ ID NOs: 3-9 or 32-40 are alsocontemplated; CDRs may be identified and such activator domains may beconstructed using well known techniques. Thus, in certain embodiments,the activator domain comprises at least one CDR of a sequence as recitedin SEQ ID NO:3-9 or 32-40. Similarly, a cytokine polypeptide modulatesactivation of the corresponding cytokine receptor, as determined in thesame fashion.

In certain embodiments, the activator domain is a growth factorpolypeptide, which binds a growth factor receptor on a cell surface.Representative such growth factor receptors are receptors for epidermalgrowth factor (EGF), Neregulin/Heregulin (NRG), fibroblast growth factor(FGF), insulin-like growth factor (e.g., IGF-I), platelet-derived growthfactor (PDGF), vascular endothelial growth factor (VEGF) and isoformsthereof (e.g., VEGF-A or VEGF-C), teratocarcinoma-derived growth factor1 (TDGF1), transforming growth factor alpha (TGF-α), transforming growthfactor beta (TGF-β) and isoforms thereof (e.g., TGF-β1 or TGF--β32),thrombopoietin (THPO) or periostin. Other such receptors includemast/stem cell growth factor receptor (SCFR), hepatocyte growth factorreceptor (HGF), ErbB-3, ErbB-4, high affinity nerve growth factorreceptor, BDNF/NT-3 growth factors receptor, NT-3 growth factorreceptor, or vascular endothelial growth factor receptor 1 (VEGFR-I).Representative cytokine receptors include, for example, FL cytokinereceptor, receptor for cytokine receptor common gamma chain,interleukin-10 receptor alpha chain, interleukin-10 receptor beta chain,interleukin-12 receptor beta-1 chain, interleukin-12 receptor beta-2chain, interleukin-13 receptor alpha-1 chain, interleukin-13 receptoralpha-2 chain, interleukin-17 receptor; interleukin-17B receptor,interleukin 21 receptor precursor, interleukin-1 receptor type I,interleukin-1 receptor type II, interleukin-2 receptor alpha chain,interleukin-2 receptor beta chain, interleukin-3 receptor alpha chain,interleukin-4 receptor alpha chain, interleukin-5 receptor alpha chain,interleukin-6 receptor alpha chain, interleukin-6 receptor beta chain,interleukin-7 receptor alpha chain, high affinity interleukin-8 receptorA, high affinity interleukin-8 receptor B, interleukin-9 receptor,interleukin-18 receptor 1, interleukin-1 receptor-like 1 precursor,interleukin-1 receptor-like 2, toll-like receptor 1, toll-like receptor2, toll-like receptor 5, CX3C chemokine receptor 1, C—X—C chemokinereceptor type 3, C—X—C chemokine receptor type 4, C—X—C chemokinereceptor type 5, C—X—C chemokine receptor type 6, C—C chemokine receptortype 1, C—C chemokine receptor type 2, C—C chemokine receptor type 3,C—C chemokine receptor type 4, C—C chemokine receptor type 6, C—Cchemokine receptor type 7 precursor, C—C chemokine receptor type 8, C—Cchemokine receptor type 9, C—C chemokine receptor type 10, C—C chemokinereceptor type 11, chemokine receptor-like 2, and chemokine XC receptor.Still other activator domains are receptors for solute carrier organicanion transporter family, member 1A2 (SLCO1A2), sphingosine kinase 1(SPHK1), secreted phosphoprotein 1 (SPP1), also called osteopontin(OPN), tumor protein 53 (TP53), troponin T type 1 (TNNT1), TSPY-likeprotein 2 (TSPYL2), visfatin, WAP four-disulfide core domain 1 (WFDC1),thymosin beta 4, wingless-type MMTV integration site family, member 11(WNT11). Representative activator domains include, for example,resistin, stromal cell-derived factor-1 (SDF-1), signal-inducedproliferation-associated gene 1 (SIPA1), and any of the other ligandslisted above, as well as portions and derivatives of the foregoing thatsubstantially retain the ability to bind to cognate receptors.

As an initial test, binding of a bi-specific fusion protein (oractivator domain thereof) to the appropriate receptor may be assessedusing techniques known in the art. In one representative assay, bindingis demonstrated by coating an appropriate solid support with therecombinant ectodomain of the appropriate receptor. An ectodomain from areceptor not recognized by the activator domain of the bi-specificfusion protein is used as a specificity control. A support substratethat does not have any immobilized receptor is also used as a control.Similar to the methods described above for binding to theischemia-associated molecule, specific, dose-dependent binding toreceptor is demonstrated using standard protocols corresponding to thesolid support and binding technology being used. In addition, studiesthat vary the amount of receptor or that include increasing levels ofsoluble target molecule as a competitor are also performed to monitorbinding and specificity. Alternatively, the bi-specific fusion proteinis immobilized to a support and the binding of the soluble ectodomain ofthe corresponding receptor(s) is used to demonstrate dose-dependent,specific binding.

The binding affinity and kinetic on and off rates for binding of thebi-specific fusion protein to the receptor(s) are also measured usingstandard techniques and compared to other negative control molecules(fusion protein with irrelevant control activator domain, fusion proteinlacking an activator domain) and positive control molecules (recombinantwild-type receptor ligand, such as a growth factor or cytokine). Theequilibrium and kinetic binding parameters of the bi-specific fusionprotein are also compared to the same parameters measured for theun-fused wild-type ligand to determine whether fusion of the ligand toother molecules affects the normal binding of the ligand to itscorresponding receptor. Such information may be used to determine theeffective dose of the bi-specific fusion protein.

A bi-specific fusion protein binds to immobilized growth factor receptoror cytokine receptor with a significantly higher affinity (e.g., atleast 100-fold) than that observed for negative controls. In addition,binding to the immobilized receptor can be competed using excess solublepolypeptide, soluble receptor, or antibodies that bind to polypeptide orreceptor and block their interaction. Preferably, the bi-specific fusionprotein binds to the growth factor or cytokine receptor with an affinitywithin 1000-fold of the native ligand binding to its receptor.

A bi-specific fusion protein (and its activator domain) further has thecapacity to mediate cognate receptor activation. Such activity may beassessed, for example, using a cellular model of ischemia reperfusion,which uses cultured cardiomyocytes such as neonatal rat ventricularmyocytes (NRVM) or cell lines. Simulated ischemia (SI) is generallyinitiated by metabolic inhibitors (deoxyglucose and dithionite) andmetabolites (high potassium, lactate, low pH) or by hypoxia in ananaerobic chamber. Reperfusion is simulated by resuspension in anoxygenated buffer. An in vitro adult cardiomyocyte pellet model ofischemia has been developed that provides the two primary components ofischemia—hypoxia and metabolite accumulation—in the absence of anyexogenous metabolic inhibitors or metabolites. Table 3 showsrepresentative methods for demonstrating the ability of a bi-specificfusion protein to prevent damage of cardiomyocytes, promote growth,motility or differentiation of cardiac stem cells and/or promote repairof damaged tissue.

TABLE 3 Activity Assessment Methods Aspect Assay Reference LocalizationDetection of activator domain Davis, Proc Natl Acad and retention incell lysate by ELISA Sci USA 103(21): kinetics of Detection of activatordomain 8155-60 (2006) activator in cells by immunofluorescence Urbanek,Proc. Natl. domain (flow cytometry or microscopic) Acad. Sci. USA 102(24): 8692-97 (2005) Signaling Detection of phospho-akt or Davis, ProcNatl Acad by activator phosphor-ERK in cells by Sci USA 103(21): domainflow cytometry, 8155-60 (2006) immunofluorescence, ELISA, Urbanek, Proc.Natl. phospho-labeling, or Western Acad. Sci. USA 102 (24): 8692-97(2005) Protection Annexin V binding by of cells immunofluorescence orflow against cytometry apoptosis Detection of caspase activity followingTUNEL-assay (reduced number hypoxia or of TUNEL-positive cells) othercell DNA laddering stressor Cell viability Enhancement of cardiomyocyteviability following exposure to H₂O₂. Number of rod-shaped cells pPCRassessment of gene expression Protection of Reduced necrotic area bycells against H&E staining necrosis Reduction in Reduction in number ofscar formation fibroblastic cells in infarct area Reduction collagendeposition Reduction in other matrix proteins associated with scarformation Migration of Time dependent increase in Urbanek, Proc. Natl.CSC into the c-kit+, sca-1+, MDR1+ cell Acad. Sci. USA 102 infarct areanumbers and numbers (24): 8692-97 (2005) undergoing transition to smallmyocytes Myocyte Frequency of distribution of Urbanek, Proc. Natl.mechanics myocyte sizes Acad. Sci. USA 102 and cell Peak shortening(24): 8692-97 (2005) fusion: Velocity of shortening and relengtheningAssessment of cell fusion (number of X chromosomes) Cardiac Comparisonof MI-treated Urbanek, Proc. Natl. functional versus MI-untreatedanimals Acad. Sci. USA 102 assessment LVEDP (24): 8692-97 (2005) LVDP+dp/dT LV Weight Chamber Volume Diastolic Wall Stress SurvivalMyocardial Composition of regenerated Urbanek, Proc. Natl. regenerationmyocardium Acad. Sci. USA 102 Assessment of BrdU+ cells in (24): 8692-97(2005) infarct area in treated versus untreated animals Myosin+ cells inthe infarct area in treated versus untreated animals Cardiac Infarctsize Urbanek, Proc. Natl. structural Fibrosis Acad. Sci. USA 102Cardiomyocyte hypertrophy (24): 8692-97(2005)

Native growth factors and cytokines can be used as activator domains. Itwill be apparent, however, that portions of such native sequences andpolypeptides having altered sequences may also be used, provided thatsuch polypeptides retain the ability to activate the cognate receptor(e.g., using one of the assays discussed below, such polypeptidesdetectably activate the receptor, and preferably activate the receptorto a degree that is at least 1% (preferably at least 10%) of thatobserved for the native ligand. Certain activator domains that bind togrowth factor receptors are provided herein in SEQ ID NOs:3-9 and 32-40.Activity of fusion proteins comprising such sequences is well known inthe art (e.g., Hashino et al., J. Biochem. 119(4):604-609 (1996); Nishiet al., Proc. Natl. Acad. Sci. USA 95:7018-23 (1998)).

An activator domain for a particular application may be selected basedon the desired therapeutic outcome. For example, an activator domainthat comprises FGF2, VEGF alpha or a portion or derivative thereof thatsubstantially retains the ability to bind to cognate receptor, maygenerally be used to increase angiogenesis. To increase survival and forstem cell differentiation (regenerative) purposes, activator domainsthat comprise IGF, HGF or NRG1 (or a portion or derivative thereof) maybe used.

In some cases, it may be desirable to assess the activity of both theactivator domain and the targeting polypeptide simultaneously. An ELISAmay be conveniently used for this purpose.

The substrate of the targeting polypeptide (e.g., DNA) is adsorbed tothe ELISA plate, which is then blocked with appropriate BSA containingbuffers. The hi-specific fusion protein is then added, followed byaddition of recombinant substrate for the activator domain (e.g., if theactivator is a growth factor, then the substrate is recombinant cognatereceptor or receptor fragment (ectodomain)). This substrate is eitherfluorescently labeled for detection or detected using a labeled antibodyto a region of the receptor that does not significantly affect ligandbinding.

The in vivo activity of the bi-specific fusion protein is generallyassessed by detecting signaling changes in molecules that are regulatedby the activator domain of the bi-specific fusion protein. Thistypically involves changes in cell surface receptor phosphorylationstatus or downstream mediators such as phospho-AKT or phospho-ERK asdetected by flow cytometry, immunofluorescence, ELISA, phospho-labeling,or Western analysis of treated tissues. Other functional assessmentsinclude tests for the number of viable cells by staining andmorphological identification, level of apoptosis by AnnexinV binding(via immunofluorescence) or flow cytometry, detection of caspaseactivity, TUNEL-assay (reduced number of TUNEL-positive cells) or DNAladdering. In each case, a bi-specific fusion protein functions in vivoif it induces a significant (e.g., at least 2-fold) change in the level,functional activity or phosphorylation of the regulated moleculedetected by the assay.

The repair of damaged tissue in a patient can be assessed using anyclinically relevant standard. For example, repair of infracted tissuecan be measured by quantitation of cell number, such as the number ofmyocytes, fibroblast, or amount of scarring, or with functional assaysfor output or structural aspects of heart function including, LVEDP,LVDP, +dp/dT, LV Weight, Chamber Volume, and Diastolic Wall Stress.Methods for such assessments are well known and amply described in theliterature. In general, a bi-specific fusion protein is said to repairdamaged tissue if it results in a significant (e.g., at least 2-fold)change in any such clinical assessment.

Polypeptide Linker

The targeting polypeptide domain and activator domain may be directlyjoined via a peptide bond. Alternatively, they may be joined via apolypeptide linker. It will be apparent that any such linker will havetwo termini, an N-terminus and a C-terminus. The linker is joined at oneterminus via a peptide bond to the targeting polypeptide domain and isjoined at the other terminus via a peptide bond to the activator domain.In certain embodiments, the linker is joined at the N-terminus to theC-terminus of the targeting polypeptide domain and at the C-terminus tothe N-terminus of the activator domain. In other embodiments, the linkeris joined at the C-terminus to the targeting polypeptide domain and atthe N-terminus to the activator domain.

Preferably, the linker is non-immunogenic in humans. More preferably,the linker is a human serum protein or a derivative thereof that retainsat least 50% sequence identity over a region that consists of at least100 consecutive amino acids. In further embodiments, the linkercomprises at least 100 consecutive amino acids that are at least 70%,80%, 85%, 90% or 95% identical to a human serum albumin amino acidsequence or a human alpha-fetoprotein amino acid sequence.Representative such linkers include those recited in any one of SEQ IDNOs:10, 12, 14-29 and 45, which may be incorporated into a bi-specificfusion protein alone or using a short (e.g., from 2 to 20 amino acidresidues) connector polypeptide at one or both ends. Suitable shortconnector polypeptides for use at the N-terminal end of the linkerinclude, for example, dipeptides such as -Gly-Ala- (GA) and -Ala-Ser-(AS). Suitable short connector polypeptides for use at the C-terminalend of the linker include, for example, dipeptides such as -Leu-Gln-(LQ)and -Thr-Gly- (TG). SEQ ID NOs:46-49 recite the linker of SEQ ID NO:45with representative connector dipeptides at both the N- and C-termini;it will be apparent, however, that such short connector polypeptides, ifpresent, may be located at either one or both termini.

Certain preferred linkers provide a prolonged half-life of thebi-specific fusion protein, as compared to fusion protein withoutlinker. The effect of a linker on half-life can be evaluated using anassay that determines stability under physiological conditions. Forexample, bi-specific fusion protein can be incubated at 37° C. in serum(e.g., human) for 120 hours, with samples removed at the start ofincubation and every 24 hours thereafter. Binding assays as describedabove are then performed to detect the level of functional bi-specificfusion protein at each time point. This level is then compared to thelevel of bi-specific fusion protein constructed without linker (or usinga different linker) to provide a half-life comparison.

Optional Elements and Representative Bi-specific Fusion Proteins

It will be apparent that elements in addition to those described abovemay optionally be included in the bi-specific fusion proteins providedherein. Such elements may be present for a variety of purposes,including to facilitate expression, preparation or purification of thebi-specific fusion protein, or to perform targeting functions. Forexample, an N-terminal leader polypeptide may be present. Representativeleader polypeptides comprise or have a sequence recited in SEQ ID NO:41or 42. A bi-specific fusion protein may also, or alternatively, comprisea polyhistadine (e.g., hexahistidine) tag to facilitate purification.Such a tag comprises at least six histidine consecutive amino acidresidues, and may be located at the C- or N-terminus. In certainembodiments, a hexahistidine tag is included at the C-terminus of thebi-specific fusion protein. Additional amino acid residues may also bepresent at the junction of the polyhistidine to the remainder of thebi-specific fusion protein. Certain bi-specific fusion proteins providedherein comprise a C-terminal polyhistidine-comprising polypeptide asrecited in SEQ ID NO:43 or 44.

Certain bi-specific fusion proteins have a general structure thatsatisfies one of the following (shown from N-terminal to C-terminal,left to right):

Or

Representative bi-specific fusion proteins comprise (from N-terminal toC-terminal):

-   -   (a) a leader polypeptide (e.g., comprising or having a sequence        recited in SEQ ID NO:41 or 42);    -   (b) a targeting polypeptide domain (e.g., comprising or having a        sequence recited in SEQ ID NO: 1, 2, 30 or 31);    -   (c) a short connector polypeptide (e.g., comprising or having        the sequence Gly-Ala- or -Ala-Ser-);    -   (d) a HSA polypeptide (e.g., comprising or having a sequence        recited in any one of SEQ ID NOs:10, 12, 14-29 and 45);    -   (e) a short connector polypeptide (e.g., comprising or having        the sequence -Leu-Gln- or -Thr-Gly-);    -   (f) an activator domain (e.g. comprising or having a sequence        recited in any one of SEQ ID NOs:3-9 and 32-40); and    -   (g) a polyhistidine-comprising polypeptide (e.g., a        hexahistadine-comprising polypeptide, such as a polypeptide        comprising or having a sequence recited in SEQ ID NO:43 or 44.

For example, certain such bi-specific fusion proteins comprise(N-terminal to C-terminal): a leader sequence as recited in SEQ ID NO:41or 42; a targeting polypeptide domain as recited in SEQ ID NO:1, 2, 30or 31; an HSA polypeptide having the sequence recited in SEQ ID NO:45; a-Gly-Ala or Ala-Ser- connector dipeptide; -Leu-Gln- or -Thr-Gly-; anactivator domain having a sequence recited in any one of SEQ ID NOs:32-40; and a hexahistidine-comprising polypeptide having a sequencerecited in SEQ ID NO:43 or 44.

Other bi-specific fusion proteins comprise (from N-terminal toC-terminal):

-   -   (a) a leader polypeptide (e.g., comprising or having a sequence        recited in SEQ ID NO:41 or 42);    -   (b) an activator domain (e.g., comprising or having a sequence        recited in any one of SEQ ID NOs:3-9 and 32-40);    -   (c) a short connector polypeptide (e.g., comprising or having        the sequence -Gly-Ala- or -Ala-Ser-);    -   (d) an HSA polypeptide (e.g., comprising or having a sequence        recited in any one of SEQ ID NOs:10, 12, 14-29 and 45);    -   (e) a short connector polypeptide (e.g., comprising or having        the sequence -Leu-Gln- or -Thr-Gly-);    -   (f) a targeting polypeptide domain (e.g., comprising or having a        sequence recited in SEQ ID NO: 1, 2, 30 or 31); and    -   (g) a poly-histidine-comprising polypeptide (e.g., comprising or        having as sequence recited in SEQ ID NO:43 or 44.

Still further bi-specific fusion proteins comprise (from N-terminal toC-terminal):

-   -   (a) a leader polypeptide (e.g., comprising or having a sequence        recited in SEQ ID NO:41 or 42);    -   (b) an activator domain (e.g., comprising or having a sequence        recited in any one of SEQ ID NOs:3-9 and 32-40);    -   (c) an HSA polypeptide that has a sequence recited in any one of        SEQ ID NOs:46-49;    -   (d) a targeting polypeptide domain (e.g., comprising or having a        sequence recited in SEQ ID NO: 1, 2, 30 or 31); and    -   (e) a poly-histidine-comprising polypeptide (e.g., comprising or        having as sequence recited in SEQ ID NO:43 or 44.        Preparation of Bi-specific Fusion Proteins

Bi-specific fusion proteins may be synthesized using standardtechniques, including liquid- and solid-phase peptide synthesis andrecombinant DNA techniques. For solid phase synthesis, the C-terminalamino acid of the sequence is attached to an insoluble support, and theremaining amino acids are added in sequence. For polypeptides longerthan about 50 amino acids, shorter regions may be synthesized in thisfashion and then condensed to form the longer polypeptide. Methods offorming peptide bonds by activation of a carboxyl terminal end (e.g., bythe use of the coupling reagent N,N′-dicyclohexylcarbodiimide) are wellknown in the art.

For recombinant DNA techniques, DNA encoding the bi-specific fusionprotein is prepared chemically or by isolating and ligating DNA encodingeach portion of the fusion protein. The DNA coding for each segment ofthe bi-specific fusion protein may be isolated from known genes orsynthesized de novo. Methods for direct chemical synthesis of DNA arewell known in the art, and such syntheses are routinely performed usingan automated synthesizer. Chemical synthesis produces a single strandedpolynucleotide, which is converted into double stranded DNA byhybridization with a complementary sequence or using DNA polymerase.While chemical synthesis of DNA is generally limited to sequences thatare shorter than the bi-specific fusion protein, it will be apparentthat the full bi-specific fusion protein may be obtained by ligation ofshorter sequences in frame. Alternatively, DNA sequences encoding thebi-specific fusion protein are prepared by cloning. Cloning techniquesare well known in the art, and are amply described, for example, bystandard references such as Sambrook et al., Molecular Cloning: ALaboratory Manual (3^(rd) ed.), Cold Spring Harbor Laboratory Press(2001). Portions of the DNA may be ligated together in frame to generatethe full length coding sequence.

Once the DNA encoding the bi-specific fusion protein is obtained, theDNA may be cloned into a vector for expression in a prokaryotic oreukaryotic host cell. Techniques for incorporating DNA into such vectorsare well known to those of ordinary skill in the art. Within such anexpression vector, the DNA encoding the bi-specific fusion protein isoperably linked to the nucleotide sequences necessary for expression(e.g., a suitable promoter and, if necessary, a terminating signal). Apromoter is a nucleotide sequence (typically located 5′ to the codingsequence) that directs the transcription of adjacently linked codingsequences. A terminating signal may be a stop codon to end translationand/or a transcription termination signal. Additional regulatoryelement(s) (e.g., enhancer elements) may also be present within anexpression vector. Such a vector is preferably a plasmid or viralvector. Preferably, an expression vector further comprises a selectablemarker, which confers resistance to a selection. This allows cells tostably integrate the vector into their chromosomes and grow to formfoci, which in turn can be cloned and expanded into cell lines. Avariety of selectable markers are known in the art, including, forexample, genes that provide resistance to ampicillin, methotrexate,mycophenolic acid, the aminoglycoside G-418, hygromycin and puromycin.Those of ordinary skill in the art are knowledgeable in the numerousexpression systems available for expression of proteins including E.coli, other bacterial hosts, yeast, and various higher eukaryotic cellssuch as the COS, CHO, HeLa and myeloma cell lines.

Host cells are transformed or transfected with the vector that comprisesthe DNA encoding the bi-specific fusion protein using standard methods.Expression in the host cell results in transcription of the DNA into thecorresponding mRNA, followed by translation of the mRNA to generate thebi-specific fusion protein.

Once expressed, the bi-specific fusion protein can be purified accordingto standard procedures, including, for example, ammonium sulfateprecipitation or affinity column chromatography. Substantially purecompositions of at least about 90 to 95% homogeneity are preferred, and98 to 99% or more homogeneity is most preferred for pharmaceutical uses.Once purified, partially or to homogeneity as desired, if to be usedtherapeutically, the polypeptides should be substantially free ofendotoxin.

Pharmaceutical Compositions

The present invention also provides pharmaceutical compositionscomprising at least one bi-specific fusion protein as described herein,together with at least one physiologically acceptable carrier. Suchcompositions may be used for treating patients who are suffering from,or at risk for, tissue damage, in order to prevent tissue damage, or torepair or regenerate damaged tissue. Such patients include, for example,patients who have experienced myocardial infarction, kidney damage,and/or ischemic stroke). If desired, other active ingredients may alsobe included within the pharmaceutical composition, such as stem cells orother agents that facilitate repair of damaged tissue.

As used herein, the term “physiologically acceptable” means approved bya regulatory agency of the Federal or a state government or listed inthe U.S. Pharmacopeia or other generally recognized pharmacopeia for usein animals, and more particularly in humans. The term “carrier” refersto a diluent, adjuvant, excipient, or vehicle with which the bi-specificfusion protein is administered. Physiologically acceptable carriers canbe sterile liquids, such as water and oils, including those ofpetroleum, animal, vegetable or synthetic origin (e.g., peanut oil,soybean oil, mineral oil, or sesame oil). Water is a preferred carrierwhen the pharmaceutical composition is administered intravenously.Saline solutions and aqueous dextrose and glycerol solutions can also beemployed as liquid carriers, particularly for injectable solutions.Suitable pharmaceutical excipients include, for example, starch,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, sodium stearate, glycerol monostearate, talc, sodium chloride,dried skim milk, glycerol, propylene, glycol, water and ethanol. Thecomposition, if desired, can also contain minor amounts of wetting oremulsifying agents, or pH buffering agents.

Pharmaceutical compositions may be formulated for any appropriate mannerof administration, including, for example, parenteral, intranasal,topical, oral, or local administration, such as by a transdermal means,for prophylactic and/or therapeutic treatment. These compositions cantake any of a variety of well known forms that suit the mode ofadministration, such as solutions, suspensions, emulsions, tablets,pills, capsules, powders, aerosols and sustained-release formulations.The composition can be formulated as a suppository, with traditionalbinders and carriers such as triglycerides. Oral formulation can includestandard carriers such as pharmaceutical grades of mannitol, lactose,starch, magnesium stearate, sodium saccharine, cellulose, magnesiumcarbonate, etc. Examples of suitable pharmaceutical modes ofadministration and carriers are described in “Remington: The Science andPractice of Pharmacy,” A. R. Gennaro, ed. Lippincott Williams & Wilkins,Philadelphia, Pa. (21^(st) ed., 2005).

Commonly, the pharmaceutical compositions provided herein areadministered parenterally (e.g., by intravenous, intramuscular, orsubcutaneous injection), or by oral ingestion or topical application.For parenteral administration, the bi-specific fusion protein can eitherbe suspended or dissolved in the carrier. A sterile aqueous carrier isgenerally preferred, such as water, buffered water, saline orphosphate-buffered saline. In addition, sterile, fixed oils may beemployed as a solvent or suspending medium. For this purpose any blandfixed oil may be employed, including synthetic mono- or diglycerides. Inaddition, fatty acids such as oleic acid find use in the preparation ofinjectible compositions. Pharmaceutically acceptable auxiliarysubstances may also be included to approximate physiological conditions,such as pH adjusting and buffering agents, tonicity adjusting agents,dispersing agents, suspending agents, wetting agents, detergents,preservatives, local anesthetics and buffering agents.

In one preferred embodiment, the pharmaceutical composition isformulated for intravenous administration to a patient (e.g., a human).Typically, compositions for intravenous administration are solutions insterile isotonic aqueous buffer. Where necessary, the composition mayalso include a solubilizing agent and a local anesthetic such aslignocaine to ease pain at the site of the injection. Generally, theingredients are supplied either separately or mixed together in unitdosage form, for example, as a dry lyophilized powder or water freeconcentrate in a sealed (e.g., hermetically sealed) container such as anampoule or sachette indicating the quantity of active agent. Where thecomposition is to be administered by infusion, it can be dispensed withan infusion bottle containing sterile pharmaceutical grade water orsaline. Where the composition is administered by injection, an ampouleof sterile water for injection or saline can be provided so that theingredients may be mixed prior to administration.

Compositions intended for oral use may be presented as, for example,tablets, troches, lozenges, aqueous or oily suspensions, dispersiblepowders or granules, emulsion, hard or soft capsules, or syrups orelixirs. Such compositions may further comprise one or more componentssuch as sweetening agents flavoring agents, coloring agents andpreserving agents. Tablets contain the active ingredient in admixturewith physiologically acceptable excipients that are suitable for themanufacture of tablets. Such excipients include, for example, inertdiluents, granulating and disintegrating agents, binding agents andlubricating agents. Formulations for oral use may also be presented ashard gelatin capsules wherein the active ingredient is mixed with aninert solid diluent, or as soft gelatin capsules wherein the activeingredient is mixed with water or an oil medium. Aqueous suspensionscomprise the active materials in admixture with one or more excipientssuitable for the manufacture of aqueous suspensions. Such excipientsinclude suspending agents and dispersing or wetting agents. Dispersiblepowders and granules suitable for preparation of an aqueous suspensionby the addition of water provide the active ingredient in admixture witha dispersing or wetting agent, suspending agent and one or morepreservatives.

Oily suspensions may be formulated by suspending the active ingredientsin a vegetable oil (e.g., arachis oil, olive oil, sesame oil or coconutoil) or in a mineral oil such as liquid paraffin. Pharmaceuticalcompositions may also be in the form of oil-in-water emulsions. The oilyphase may be a vegetable oil or a mineral oil or mixture thereof.Suitable emulsifying agents include, for example, naturally-occurringgums, naturally-occurring phosphatides and anhydrides.

Pharmaceutical compositions may be sterilized by conventionalsterilization techniques, or may be sterile filtered. Sterile aqueoussolutions may be packaged for use as is, or lyophilized, the lyophilizedpreparation being combined with a sterile aqueous carrier prior toadministration. The pH of an aqueous pharmaceutical compositiontypically will be between 3 and 11, more preferably between 5 and 9 orbetween 6 and 8, and most preferably between 7 and 8, such as 7 to 7.5.

Bi-specific fusion proteins provided herein are generally present withina pharmaceutical composition at a concentration such that administrationof a single dose to a patient delivers a therapeutically effectiveamount. A therapeutically effective amount is an amount that results ina discernible patient benefit, such as detectable repair or regenerationof damaged tissue or diminution of symptoms of tissue damage.Therapeutically effective amounts can be approximated from the amountssufficient to achieve detectable tissue repair or regeneration in one ormore animal models exemplified in Table 3. Nonetheless, it will beapparent that a variety of factors will affect the therapeuticallyeffective amount, including the activity of the bi-specific fusionprotein employed; the age, body weight, general health, sex and diet ofthe patient; the time and route of administration; the rate ofexcretion; any simultaneous treatment, such as a drug combination; andthe type and severity of the tissue damage in the patient undergoingtreatment. Optimal dosages may be established using routine testing, andprocedures that are well known in the art. Dosages generally range fromabout 0.5 mg to about 400 mg of bi-specific fusion protein per dose(e.g., 10 mg, 50 mg, 100 mg, 200 mg, 300 mg, or 400 mg per dose). Ingeneral, compositions providing dosage levels ranging from about 0.1 gto about 100 g per kilogram of body weight per day are preferred. Incertain embodiments, dosage unit forms contain between from about 10 gto about 100 g of bi-specific fusion protein.

Pharmaceutical compositions may be packaged for treating or preventingtissue damage (e.g., for treatment of myocardial infarction or kidneydamage). Packaged pharmaceutical preparations include a containerholding a therapeutically effective amount of at least onepharmaceutical composition as described herein and instructions (e.g.,labeling) indicating that the contained composition is to be used fortreating tissue damage (such as myocardial infarction or kidney damage)in a patient. Pharmaceutical compositions may be packaged in multiplesingle dose units, each containing a fixed amount of bi-specific fusionprotein in a sealed package. Alternatively, the container may holdmultiple doses of the pharmaceutical composition.

Methods of Treatment

The pharmaceutical compositions can be administered to a patient(preferably a mammal such as a cow, pig, horse, chicken, cat, dog, ormore preferably a human) to treat pathological tissue damage in thepatient. Within the context of the present invention, the term“treatment” encompasses both prophylactic and therapeuticadministration. In prophylactic applications, a pharmaceuticalcomposition as described herein is administered to a patient susceptibleto or otherwise at risk for developing pathological tissue damage, inorder to prevent, delay or reduce the severity of tissue damage. Intherapeutic applications, treatment is performed in order to reduce theseverity of the pathological tissue damage exist in the patient prior totreatment. Representative pathological tissue damage includes hearttissue damage (e.g., damage associated with myocardial infarction),kidney tissue damage and ischemic stroke.

Any of a variety of known delivery systems can be used to administer abi-specific fusion protein including, for example, encapsulation inliposomes, microparticles, microcapsules, recombinant cells capable ofexpressing the bi-specific fusion protein, receptor-mediated, or aretroviral or other nucleic acid vector. The bi-specific fusion proteinmay be administered by any convenient route, for example by infusion orbolus injection, by absorption through epithelial or mucocutaneouslinings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and maybe administered together with other biologically active agents.Administration can be systemic or local. In addition, it may bedesirable to introduce the bi-specific fusion protein into the centralnervous system by any suitable route, including intraventricular andintrathecal injection; intraventricular injection may be facilitated byan intraventricular catheter, for example, attached to a reservoir, suchas an Ommaya reservoir. Pulmonary administration can also be employed,e.g., by use of an inhaler or nebulizer, and formulation with anaerosolizing agent.

In a specific embodiment, it may be desirable to administer the bsBAs ofthe invention locally to the area in need of treatment; this may beachieved by, for example, local infusion during surgery, topicalapplication (e.g., in conjunction with a wound dressing after surgery),by injection, by means of a catheter, by means of a suppository, or bymeans of an implant, said implant being of a porous, non-porous, orgelatinous material, including membranes, such as sialastic membranes,or fibers. In another embodiment, a vesicle, such as a liposome, can beused to deliver the bi-specific fusion protein. In yet anotherembodiment, the bi-specific fusion protein is delivered in a controlledrelease system; for example, such a controlled release system may beplaced at or near the therapeutic target (e.g., an organ of the bodythat has experienced or is at risk for tissue damage). The use of suchdelivery systems is well known to those of ordinary skill in the art.

Without wishing to be bound by any particular theory, it is believe thatthe bi-specific fusion proteins provided herein are effective fortreating pathological tissue damage at least in part due to theirability to recruit stem cells to the damaged tissue. In certain cases,sufficient stem cells may reside within the patient (e.g., residentcardiac stem cells). In certain embodiments, however, it may bebeneficial to co-administer stem cells (e.g., bone marrow-derivedautologous stem cells). Such stem cells may be administered before orafter the bi-specific fusion protein, or may be administeredsimultaneously (either in the same pharmaceutical composition or inseparate compositions).

As noted above, the optimal dose depends on certain factors known in theart, but generally ranges from about 0.5 mg to about 400 mg ofbi-specific fusion protein per dose (e.g., 10 mg, 50 mg, 100 mg, 200 mg,300 mg, or 400 mg per dose). A dose of bi-specific fusion protein(within a pharmaceutical composition as described above) can beadministered therapeutically to a patient one or more times per hour,day, week, month, or year (e.g., 2, 4, 5, 6, 7, 8, 9, 10, 11, or 12times per hour, day, week, month, or year). More commonly, a single doseper day or per week comprising an amount of bi-specific fusion proteinranging from about 0.1 g to about 100 g per kilogram of body weight isadministered.

In other embodiments, a pharmaceutical composition comprising abi-specific fusion protein may be administered to a patient in a dosagethat ranges from about 0.5 mg per week to about 400 mg per week, about1.0 mg per week to about 300 mg per week, about 5 mg per week to about200 mg per week, about 10 mg per week to about 100 mg per week, about 20mg per week to about 80 mg per week, about 100 mg per week to about 300mg per week, or about 100 mg per week to about 200 mg per week.Alternatively, a pharmaceutical composition comprising a bi-specificfusion protein may be administered at a dose that ranges from about 0.5mg every other day to about 100 mg every other day, about 5 mg everyother day to about 75 mg every other day, about 10 mg every other day toabout 50 mg every other day, or about 20 mg every other day to about 40mg every other day. A pharmaceutical composition comprising abi-specific fusion protein may alternatively be administered at a dosethat ranges from about 0.5 mg three times per week to about 100 mg threetimes per week, about 5 mg three times per week to about 75 mg threetimes per week, about 10 mg three times per week to about 50 mg threetimes per week, or about 20 mg three times per week to about 40 mg threetimes per week.

In further embodiments of, a pharmaceutical composition comprising abi-specific fusion protein is administered to a mammal (e.g., a human)continuously for 1, 2, 3, or 4 hours; 1, 2, 3, or 4 times a day; everyother day or every third, fourth, fifth, or sixth day; 1, 2, 3, 4, 5, 6,7, 8, 9, or 10 times a week; biweekly; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, or 30 times a month; bimonthly; 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10times every six months; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20 times a year; or biannually. It will beapparent that a pharmaceutical composition comprising a bi-specificfusion protein may, but need not, be administered at differentfrequencies during a therapeutic regime.

The following Examples are offered by way of illustration and not by wayof limitation. Unless otherwise specified, all reagents and solvents areof standard commercial grade and are used without further purification.Using routine modifications, the procedures provided in the followingExamples may be varied by those of ordinary skill in the art to make anduse other bi-specific fusion proteins and pharmaceutical compositionswithin the scope of the present invention.

EXAMPLES Example I Preparation of a Representative Bi-Specific FusionProtein

A bi-specific fusion protein in which targeting polypeptide domain bindsto DNA and the activator domain is NRG1 is prepared. The two domains arejoined by a modified human serum albumin (HSA) linker. The NRG1 isrecombinantly fused to the amino terminus of the HSA linkerincorporating a short connector polypeptide and the anti-DNA scFv isrecombinantly fused to the carboxy terminus of the modified HSA linkerincorporating an additional short connector polypeptide. The modifiedHSA linker contains two amino acid substitutions. A cysteine residue atposition 34 of native HSA is mutated to serine in order to reducepotential protein heterogeneity due to oxidation at this site. Anasparagine residue at amino acid 503 of native HSA, which may besensitive to deamidation, resulting in decreased pharmacologichalf-life, is mutated to glutamine. The modified HSA linker confers anextended circulating half-life on the bi-specific fusion protein.

Example II In Vitro Activity of a Bi-Specific Fusion Protein

The activity of both components of the representative bi-specific fusionprotein prepared in Example 1 (in which the targeting polypeptide domainbinds to DNA and the activator domain is NRG1) are tested using an ELISAdesigned to give activity only when both arms of the bi-specific fusionprotein are bound to their substrates simultaneously. The ELISA isperformed essentially as described in Stokes et al., J. Clin. Pathol.35(5): 566-573 (1982) and Gripenberg et al., Scand. J. Immunol.1:151-157 (1978). More specifically, 1 to 50 ng/ml solution of thebi-specific fusion protein in PBS is added to the wells of a platepre-adsorbed with DNA (Anti-DS-DNA antibody ELISA kit (Alpha DiagnosticInternational, Dist by AutogenBioclear, UK) and incubated and washedaccording to manufacturer's directions until the step in which thedetection antibody is added. At this stage, 100 μl of 1-50 ng/mlsolution of Biotinylated goat anti-human NRG1-131 (R&D Systems BAF377)(antibody to the ‘activator arm’) in PBS/1% BSA/0.05% Tween is added toall wells and incubated for 1 hr at room temperature, washed in PBS with0.05% Tween-20. 100 μl of Streptavidin-HRP (1:200 dilutions of stock 2ug/ml, (R&D Systems 890803)) diluted in PBS is added to each well andincubated 30 min at room temperature. After a final wash in PBS with0.05% Tween-20, 100 μA of SuperSignal ELISA Pico ChemiluminescentSubstrate (as per manufacturer's instructions, Pierce, cat#34077) isadded and luminescence (representative of positive signal) is measuredon Fusion Microplate reader (Packard) or similar instrument.

The amount of signal detected is significantly higher (at least 100-foldhigher) in the wells with bi-specific fusion protein than in wellswithout DNA or negative controls that contain a dead arm (i.e., does notcontain an activator domain or targeting polypeptide domain). Inaddition, the signal is seen to vary with the amount of bi-specificfusion protein added to the wells.

Example III In Vivo Activity of a Bi-Specific Fusion Protein

The in vivo activity of the representative bi-specific fusion proteinprepared in Example 1 is determined by detecting signaling changes in amolecule that is regulated by the activator domain of the fusionprotein. For the activator domain in this fusion protein NRG1, activityis assessed by detection of increased phosphorylated ErbB-3 in cells ofhearts treated with the bi-specific fusion compared to untreated or mocktreated hearts. Myocardial infarction is generated in C57BL/6 mice byligation of the left coronary artery (LCA) following endotrachealintubation, ventilation and thoracotomy. Coronary occlusion is confirmedby acute inspection of color change of the left ventricle wall, and STelevation on the electrocardiogram before chest closure. Sham-operatedmice undergo the same surgical procedure without LCA ligation.

Hearts from normal mice or those following induction of myocardialinfarction, from both control and bi-specific fusion protein treatedmice, are removed, fixed in 4% paraformaldehyde, embedded, sectioned andmounted as described in Dhein, Mohr and Delmar, Practical Methods inCardiovascular Research, 2005, p. 473 (Springer, New York).Phospho-ErbB3 antibody (Cell Signaling Technology; Beverly, Mass.) isused for detection of Phospho-ErbB3 by immunofluorescence. A 2-foldincrease or more in phospho-ErbB3 levels in treated versus untreatedhearts is observed and is indicative of functional activator. Theincrease is in either the number (number per field, or percentage oftotal) of cells exhibiting signal, the intensity of signal per cell, orboth.

Example IV Tissue Damage Repair in Mice Using a Bi-Specific FusionProtein

A composition comprising the representative bi-specific fusion proteinof Example 1 is administered to a mouse following myocardial infarction,induced as described above. Administration is via intravenous injection(e.g., tail vein). Following administration, heart function is assessedas follows. Mice are anesthetized with chloral hydrate (400 mg/kg bodyweight, i.p.), and the right carotid artery is cannulated with amicrotip pressure transducer (model SPR-671, Millar) for themeasurements of left ventricular (LV) pressures and LV+ and −dP/dt inthe closed-chest preparation. Measurements are compared to thoseobtained from untreated control mice to confirm that treatment with thebi-specific fusion protein affects heart function. A significantimprovement is observed in heart function as assessed using at least oneof these measurements.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications of changesin light thereof are to be included within the spirit and purview ofthis application and scope of the appended claims. All publication,patents and patent applications cited herein are hereby incorporated byreference in their entirety for all purposes.

The invention claimed is:
 1. A bi-specific fusion protein comprising:(a) a targeting domain consisting of an amino acid sequence of SEQ ID NO31; and (b) an activator domain consisting of an amino acid sequence ofSEQ ID NO
 3. 2. The bi-specific fusion protein of claim 1 furthercomprising a linker.
 3. The bi-specific fusion protein of claim 2wherein the linker is a non-immunogenic protein.
 4. The bi-specificfusion protein of claim 2, wherein the linker comprises SEQ ID NO
 10. 5.The bi-specific protein of claim 1 or claim 2 further comprising aleader polypeptide.
 6. The bi-specific protein of claim 5 wherein theleader polypeptide consists of an amino acid sequence of SEQ ID NO: 41or SEQ ID NO
 42. 7. The bi-specific fusion protein of claim 1 whereinthe targeting domain is at the amino terminus of the activator domain orwherein the targeting domain is at the carboxy terminus of the activatordomain.
 8. The bi-specific fusion protein of claim 1 consisting of anamino acid sequence selected from the group consisting of: (a) SEQ IDNOs 42, 3, 10 and 31, wherein the amino acid sequence of SEQ ID NO 3 isfused to the C terminus of the amino acid sequence of SEQ ID NO 42 andto the N-terminus of the amino acid sequence of SEQ ID NO 10 and whereinthe amino acid sequence of SEQ ID NO 10 is fused to the N-terminus ofthe amino acid sequence of SEQ ID NO 31, (b) SEQ ID NOs 42, 31, 10 and3, wherein the amino acid sequence of SEQ ID NO 31 is fused to the Cterminus of the amino acid sequence of SEQ ID NO 42 and to theN-terminus of the amino acid sequence of SEQ ID NO 10 and wherein theamino acid sequence of SEQ ID NO 10 is fused to the N-terminus of theamino acid sequence of SEQ ID NO 3, (c) SEQ ID NOs 42, 3 and 31, whereinthe amino acid sequence of SEQ ID NO 3 is fused to the C terminus of theamino acid sequence of SEQ ID NO 42 and to the N-terminus of the aminoacid sequence of SEQ ID NO 31, (d) SEQ ID NOs 3, 10 and 31, wherein theamino acid sequence of SEQ ID NO 10 is fused to the C terminus of theamino acid sequence of SEQ ID NO 3 and to the N-terminus of the aminoacid sequence of SEQ ID NO 31, (e) SEQ ID NOs 42, 31 and 3, wherein theamino acid sequence of SEQ ID NO 31 is fused to the C terminus of theamino acid sequence of SEQ ID NO 42 and to the N-terminus of the aminoacid sequence of SEQ ID NO 3, (f) SEQ ID NOs 31, 10 and 3, wherein theamino acid sequence of SEQ ID NO 10 is fused to the C terminus of theamino acid sequence of SEQ ID NO 31 and to the N-terminus of the aminoacid sequence of SEQ ID NO 3, (g) SEQ ID NOs 41, 3, 10, 31, wherein theamino acid sequence of SEQ ID NO 3 is fused to the C terminus of theamino acid sequence of SEQ ID NO 41 and to the N-terminus of the aminoacid sequence of SEQ ID NO 10 and wherein the amino acid sequence of SEQID NO 10 is fused to the N-terminus of the amino acid sequence of SEQ IDNO 31, (h) SEQ ID NOs 41, 31, 10 and 3, wherein the amino acid sequenceof SEQ ID NO 31 is fused to the C terminus of the amino acid sequence ofSEQ ID NO 41 and to the N-terminus of the amino acid sequence of SEQ IDNO 10 and wherein the amino acid sequence of SEQ ID NO 10 is fused tothe N-terminus of the amino acid sequence of SEQ ID NO 3, (i) SEQ ID NOs41, 3, and 31, wherein the amino acid sequence of SEQ ID NO 3 is fusedto the C terminus of the amino acid sequence of SEQ ID NO 41 and to theN-terminus of the amino acid sequence of SEQ ID NO 31, and (j) SEQ IDNOs 41, 31 and 3, wherein the amino acid sequence of SEQ ID NO 31 isfused to the C terminus of the amino acid sequence of SEQ ID NO 41 andto the N-terminus of the amino acid sequence of SEQ ID NO
 3. 9. Abi-specific fusion protein comprising: (a) a targeting domain consistingof an amino acid sequence of SEQ ID NO 31, (b) an activator domainconsisting of an amino acid sequence of SEQ ID NO 3; and (c) apolypeptide linker consisting of an amino acid sequence of SEQ ID NO 10.